Orthopoxvirus vectors, genes and products thereof

ABSTRACT

An orthopoxvirus vector, such as vaccinia, is described in which the A52R protein from vaccinia, or a closely related protein from any orthopoxvirus is not expressed or is expressed but is non-functional. Also described is the use of a vaccinia virus A52R protein or a closely related protein from any orthopoxvirus, or a functional peptide, peptidometic, fragment or derivative thereof, or a DNA vector expressing any of the above in the modulation and/or inhibition of IL1R/TLR superfamily signalling.

FIELD OF THE INVENTION

The invention relates to a viral protein that is a novel inhibitor ofthe immunologically important transcription factor Nuclear factor kappaB (NFκB). The invention also relates to the mechanism whereby theinhibitor functions, and the use of the inhibitor, or informationderived from its mechanism of action, in designing peptides or smallmolecule inhibitors for use in NFκB related diseases and conditions. Theinvention also relates to a recombinant vaccinia virus (VV) as a vaccinecandidate for the prevention of smallpox or other infectious diseases,or for the prevention or treatment of cancer.

BACKGROUND

Members of the IL-1 receptor/Toll-like receptor (IL-1R/TLR) superfamilyare key mediators in innate and adaptive immunity (Akira, S., Takeda, K.& Kaisho, T. Nature Immunol. 2, 675-680 (2001)). The superfamily isdefined by the presence of a cytosolic motif termed the Toll/IL-1receptor (TIR) domain. The family includes receptors for theproinflammatory cytokines IL-1 and IL-18 as well as the TLR members,which participate in the recognition of pathogens by responding topathogen associated molecular patterns (PAMPs) and activating signallingpathways leading to altered gene expression (Bowie, A. & O'Neill, L. A.J. J. Leuk. Biol. 67, 508-514 (2000)). The TLRs were discovered on thebasis of their amino acid similarity to Toll, a Drosophilia proteininvolved in mediating antifungal defence (Lemaitre, B., Nicolas, E.,Michaut, L., Reichart, J. & Hoffmann, J. Cell 86, 973-983 (1996)). Tenmammalian TLRs have been identified to date. TLR4, TLR5 and TLR9 areessential in the respective recognition of lipopolysaccharide (LPS),bacterial flagellin and unmethylated CpG motifs which are present inbacterial DNA (Poltorak, A. et al. i Science 282, 2085-2088 (1998);Qureshi, S. T. et al. J. Exp. Med. 189, 615-625 (1999); Hayashi, F. etal. Nature 410, 1099-1103 (2001); Hemmi, H. et al. Nature 408, 740-745(2000)). TLR2 recognises bacterial lipoproteins and other Gram-positivemolecular patterns, but only when present as a heterodimer incombination with either TLR1 or TLR6 (Brightbill, H. D. et al. Science285, 732-736 (1999); Aliprantis, A et al. Science 285, 736-739 (1999);Underhill, D. et al., Nature 401, 811-815 (1999); Takeuchi, O. et al.Immunity 11, 443-451 (1999); Ozinsky, A. et al. Proc Natl. Acad. Sci.USA 97, 13766-13771 (2000); Takeuchi, O. et al. Int. Immunol. 13,933-940 (2001)). TLRs have also been implicated in sensing viralinfections. TLR4 has been shown to be necessary for thecytokine-stimulating ability of F protein from respiratory syncytialvirus (RSV) and also for murine retrovirus activation of B cells(Kurt-Jones, E. A. et al. Nature Immunol 1, 398-401 (2000); Rassa, J.C., Meyers, J. L., Zhang, Y., Kudaravalli, R & Ross, S. Proc. Natl.Acad. Sci. USA 99, 2281-2286 (2002)). TLR3 meanwhile was identified as areceptor activated in response to poly(I:C), a synthetic double-strandedRNA (dsRNA) mimic of viral dsRNA. Poly(I:C) activation of cells via TLR3led to the activation of the transcription factor NFκB and theproduction of type I interferons, which are important in anti-viralinnate immunity (Alexopoulou, L., Czopik-Holt, A., Medzhitov, R. &Flavell, R. Nature 413, 696-712 (2001)). Further, imidazoquinolinecompounds known to have potent anti-viral properties activated immunecells via TLR7 (Hemmi, H. et al. Nature Immunol. 3, 196-200 (2002)).

Since these receptors all contain the signalling TIR domain, stimulationof all the family members with the appropriate ligands leads toactivation of NFκB and also the mitogen-activated protein kinases(MAPKs), p38, JNK and p42/44. NFκB is a homo- or hetero-dimer of membersof the Rel family of transcriptional activators that is involved in theinducible expression of a wide variety of important cellular genes. Theactivation of NFκB by IL-1, IL-18, TLR2, TLR7 and TLR9 is absolutelydependent on the cytoplasmic TIR domain-containing protein MyD88 (Hemmi,H. et al. Nature Immunol. 3, 196-200 (2002); Adachi, O. et al. Immunity9, 143-150 (1998); Takeuchi, O. et al. J. Immunol 164, 554-557 (2000);Schnare, M., Holt, A. C., Takeda, K., Akira, S. & Medzhitov, R. Curr.Biol. 10, 1139-1142 (2000)), which is recruited to receptor TIR domains(Medzhitov, R. et al. Mol. Cell 2, 253-258 (1998); Wesche, H., Henzel,W. J., Shilinglaw, W., Li, S. & Cao, Z. Immunity 7, 837-847 (1997);Muzio, M., Ni, J., Feng, P. & Dixit, V. M. Science 278, 1612-1615(1997)). However TLR4 is able to activate NFκB, by both aMyD88-dependent and MyD88-independent pathway, while NF B activation byTLR3 is completely MyD88-independent (Alexopoulou, L., Czopik-Holt, A.,Medzhitov, R. & Flaveli, R. Nature 413, 696-712 (2001); Kawai, T.,Adachi, O., Ogawa, T., Takeda, K. & Akira, S. Immunity 11,115-122(1999)). The MyD88 dependent pathway is involved in TNF induction by LPSin dendritic cells whereas the MyD88 independent pathway leads to theupregulation of costimulatory molecules required for dendritic cellmaturation, and induction of genes dependent on the transcription factorInterferon Regulatory Factor 3 (IRF3) (Kaisho, T., Takeuchi, O., Kawai,T., Hoshino, K. & Akira, S. J. Immunol, 166, 5688-5694 (2001)). Animportant example of such a gene is Interferon-(IFN). For TLR4 and TLR2,another TIR adapter molecule, MyD88Adaptor-Like (Mal, also known asTIRAP) is involved in the MyD88 dependent pathway (Fitzgerald, K. A. etat. Nature 413, 78-83 (2001); Horng, T., Barton, G. M. & Medzhitov, R.Nature Immunol, 2, 835-841 (2001); Yamamoto, M. et al. Nature 420,324-329 (2002); Horng, T. et al. Nature 420, 329-333 (2002)). Activationof NFκB by the MyD88 dependent pathway can proceed via recruitment byMyD88 of IL-1 receptor-associated kinase (IRAK) and/or IRAK2, while Malfunctions via the recruitment of IRAK2 (Fitzgerald, K. A. et al. Nature413, 78-83 (2001)). IRAK or IRAK2 activation in turn leads torecruitment of tumor necrosis factor receptor-associated factor 6(TRAF6). TRAF6 is required for the ubiquitination and activation of thekinase TAK-1, which, in complex with TAB1, phosphorylates IκB kinase(IKK) leading to NFκB activation (Wang, C. et al. Nature 412, 346-351(2001)). Recently another TIR adapter termed TICAM-1 or TRIF has beendiscovered (Yamamoto, M. et al. J. Immunol. 169, 6668-6672 (2002);Oshiumi, H. et al. Nature Immunol 4, 161-167 (2003)). It has been shownthat for TLR4, TRIF mediates the MyD88-independent pathway to IRF3,while for TLR3, TRIP mediates both NFB a n d IRF3 activation (Hoebe, K.et al. Nature doi:10.1038/nature01889 (2003); Yamamoto, M. et al.Science doi:10.1126/science.1087262 (2003)).

Methods to inhibit key components in the activation pathway of NFκBwould have valuable therapeutic application.

STATEMENTS OF INVENTION

According to the invention there is provided an orthopoxvirus vector,such as vaccinia, wherein the A52R protein from vaccinia, or a closelyrelated protein from any orthopoxvirus is not expressed or is expressedbut is non-functional.

In one embodiment part or all of the nucleotide sequence encoding A52Ris deleted from the viral genome.

In another embodiment the nucleotide sequence encoding A52R isinactivated by mutation or the insertion of foreign DNA.

The nucleotide sequence encoding A52R may be changed.

In one embodiment the A52 gene comprises amino acid SEQ ID No. 1.

The orthopoxvirus vector of the invention preferably has enhancedimmunogenicity and/or safety compared to the wild type orthopoxvirus.

The invention also provides a medicament comprising an orthopoxvirusvector of the invention.

In another aspect the invention provides a vaccine comprising anorthopoxvirus vector of the invention.

In another aspect the invention provides a recombinant orthopoxvirusincapable of expressing a native A52R protein. A vaccine may comprisesuch a recombinant virus.

In a further aspect the invention provides a method of attenuating anorthopoxvirus vector such as vaccinia virus, comprising the steps of:

-   -   (a) deleting part or all of the nucleotide sequence encoding        A52R from the viral genome; and/or    -   (b) inactivating one or more of said nucleotide sequence by        mutating said nucleotide sequence or by inserting foreign DNA;        and/or    -   (c) changing said nucleotide sequence to alter the function of a        protein product encoded by said nucleotide sequence.

In one embodiment the invention provides a method of inhibiting IL1R/TLRsuperfamily signalling comprising administering an effective amount ofvaccinia A52R protein, or a closely related protein from anyorthopoxvirus or a functional peptide, peptidometic fragment orderivative thereof or a DNA vector capable of expressing such a proteinor fragment thereof.

In another embodiment the invention provides a method of modulatinganti-viral immunity in a host comprising administering an orthopoxvirusvector such as vaccinia virus of the invention or a functional peptide,peptidometic, fragment or derivative thereof.

The invention also provides an immunogen comprising an orthopoxvirusvector, such as vaccinia virus of the invention or a recombinant virusvector.

In another aspect the invention provides use of a vaccinia virus A52Rprotein or a closely related protein from any orthopoxvirus, or afunctional peptide, peptidometic, fragment or derivative thereof, or aDNA vector expressing any of the above in the modulation and/orinhibition of IL1R/TLR superfamily signalling.

The use may be in the modulation and/or inhibition of IL1R/TLRsuperfamily induced NFκB activation.

The use may be in the modulation of IL1R/TLR superfamily induced MAPkinase activation.

The use may be in the modulation or inhibition of TLR induced IRF3activation.

In one aspect the vaccinia virus A52R protein, or a closely relatedprotein from any orthopoxvirus, inhibits Toll-like receptor proteins.

The use may be in the modulation and/or inhibition of NF-κB activity byinteraction of A52R with TRAF6. The A52R protein may inhibit formationof an endogenous signalling complex containing TRAF6/TAB1.

The use may be in the modulation and/or inhibition of NF-κB activity byinteraction of A52R with IRAK2.

The A52R protein may inhibit Mal/IRAK2 interaction.

The invention further provides a viral protein comprising amino acid SEQID No. 2.

In another aspect the invention provides use of a viral protein or afunctional peptide, peptidometic, fragment or derivative thereof in themodulation and/or inhibition of IL1R/TLR superfamily signalling. The usemay be in the modulation and/or inhibition of IL1R/TLR superfamilyinduced NFκB activation. The use may be in the inhibition of IL1R/TLRsuperfamily induced p38 MAP kinase activation.

In one embodiment the said truncated vaccinia virus A52R proteininhibits Toll-like receptor proteins.

In another aspect the invention provides the use of the viral protein inthe modulation and/or inhibition of NF-κB activity by interaction of thesaid truncated A52R with IRAK2.

According to the invention there is provided a peptide derived from,and/or a small molecule inhibitor designed based on a viral proteincomprising amino acid SEQ ID No. 1 or SEQ ID No. 2.

The invention also provides a method of screening compounds thatmodulate the NF-κB and/or p38 MAP kinase related pathway comprisingmeasuring the effect of a test compound on the interaction of A52R or aviral protein fragment comprising amino acid SEQ ID No. 2 or afunctional peptide, peptidometic, fragment or derivative thereof withTRAF6 and/or IRAK2.

In another aspect the invention provides a method of identifyingsignalling pathways that require TRAF6 and/or IRAK2, comprisingmeasuring their sensitivity to A52R or a viral protein comprising aminoacid SEQ ID No. 2.

The invention further provides use of a functional peptide,peptidometic, or fragment derived from vaccinia virus A52R protein, orany closely related orthopoxvirus protein, or a small molecule inhibitordesigned based on A52R in the treatment and/or prophylaxis of IL-1R/TLRsuperfamily-induced NF-κB or p38 MAP kinase related diseases orconditions. The NF-κB related disease or condition mall be selected fromany one or more of a chronic inflammatory disease, allograft rejection,tissue damage during insult and injury, septic shock and cardiacinflammation, autoimmune disease, cystic fibrosis or any diseaseinvolving the blocking of Thl responses. The chronic inflammatorydisease may include any one or more of RA, asthma or inflammatory boweldisease. The autoimmune disease may be systemic lupus erythematosus.

The use may be in treatment and/or prophylaxis of inflammatory disease,infectious disease or cancer.

The protein may be derived from an orthopoxvirus.

The term functional peptide, peptidometic, fragment or derivative asused herein are understood to mean any molecule or macromoleculeconsisting of a portion of the A52R protein, or designed using sequenceor structural information from A52R.

The term non-functional is understood to mean not functioning in thenormal way compared to how the wild-type A52R protein would function.

The term ‘closely related’ is understood to mean ‘greater than 50% aminoacid identity’.

The invention is in the field of poxviruses. The family name ispoxvirus, the subfamily name is chordopoxyirinae (infect vertebrates)and the genus is orthopoxvirus which includes species of virus some ofwhich have A52R homologs. The best known species of this genus arevaccinia, variola, camelpox, cowpox, monkeypox and ectromelia (infectsmice).

The invention relates to any orthopoxvirus vector in which A52R protein,is deleted/modified.

The invention also relates to the use of A52R protein from anyorthopoxvirus.

The invention further relates to the use of a DNA vector expressing A52Rprotein.

BRIEF DESCRIPTION OF THE DRAWING

The invention will be more clearly understood from the followingdescription thereof given by way of example only with reference to theaccompanying drawings in which:—

FIGS. 1 a to c are graphs showing the inhibition by A52R of theactivation of NF % B by multiple TLR family members;

FIGS. 2 a and b are graphs showing the inhibition by A52R of theactivation of NFκB and the IFNβ promoter by TLR agonists in the murinemacrophage cell line RAW264.7;

FIGS. 3 a to f are immuno-blots showing the immunoprecipitation of A52Rwith TRAF6 and IRAK2 but not with other TLR signalling components;

FIGS. 4 a and b are immuno-blots showing immunoprecipitation of A52Rwith endogenous TRAF6, and with the TRAF6 TRAP domain, but not withTRAF2;

FIG. 5 is an immuno-blot showing the different effects of A52R on aTRAF6-TAB1-containing signalling complex and a TAB1-TAK complex;

FIGS. 6 a to d show characterisation and functional consequences of theinteraction of A52R with IRAK2;

FIGS. 7 a and b show that a truncated version of A52R, ΔA52R, whichlacks amino acids VDVWRNEKLFSRWKYCLRAKILFINDHMLDKIKSILQNRLVYVEMS at theC-terminal, interacts with IRAK2 but not TRAF6;

FIGS. 8 a and b show that ΔA52R can inhibit IL-1 and TLR4 mediated NFκBactivation;

FIGS. 9 a and b show that both A52R and A52R can inhibit TRIF-dependentsignals;

FIGS. 10 a to c show differences in the ability of A52R and ΔA52R toactivate and inhibit p38 MAP kinase; and

FIGS. 11 a and b are graphs showing that deletion of A52R from thevaccinia virus genome attenuates the virus, as measured by weight lossand signs of illness of mice that are infected intranasally.

DETAILED DESCRIPTION

Poxviruses are a family of complex DNA viruses that include variolavirus, the causative agent of smallpox, and the antigenically relatedvirus used to eradicate this disease, vaccinia virus (VV).Orthopoxviruses such as VV display unique strategies for the evasion ofhost immune responses such as the ability to produce secreted decoyreceptors for cytokines such as IL-1, TNF, and the interferons IFNαβ andIFNγ.

The present invention concerns a VV protein A52R, which is known to bean intracellular inhibitor of signalling by the IL-1R/TLR superfamily.A52R has been shown to inhibit IL-1R-, IL-18R- and TLR4-induced NFκBactivation (Bowie, A. et al. Proc. Natl. Acad. Sci. USA 97, 10162-10167(2000)). In the present invention it was surprisingly found that A52Rcan in fact inhibit NFκB induction by multiple TLRs. It was found thatA52R inhibits numerous other TLR pathways to NFκB activation, namelyTLR2&6, TLR2&1, TLR5 and TLR3-dependent poly(I:C) (FIGS. 1 and 2).Inhibition was due to the ability of A52R to associate with both TRAF6and IRAK2 FIGS. 3 and 4) and hence disrupt signalling complexes requiredfor IL1R/TLR-induced NFκB activation (FIGS. 5 and 6). Furthermore, A52Rwas shown to also be capable of antagonising induction of theIFN-dependent, MyD88-independent pathway, triggered by TLR3 and TLR4(FIGS. 2 a and 9). A truncated version of A52R, which retained theability to target IRAK2 (FIG. 7), was a more potent inhibitor of TIRsignalling than A52R (FIGS. 8 to 10). Importantly, a deletion mutantvirus lacking the A52R gene was shown to be attenuated compared to wildtype and revertant controls in vivo (FIG. 11).

There is intense interest in the IL-1R/TLR family at present, given itsemerging central importance in the innate immune response to diversepathogens (Akira, S., Takeda, K. & Kaisho, T. Nature Immunol. 2, 675-680(2001)). During the course of viral infection the body mounts severallines of host defence involving constituents of the IL-1R/TLRsuperfamily. The cytokines IL-1 and IL-18 are key regulators of theinnate and adaptive immune response to viral infection. In particularIL-1 is responsible for inducing a fever response during viralinfection, which is antagonized by the production of a soluble IL-1binding protein (B15R) by VV (Alcami, A. & Smith, G. L. Cell 71, 153-167(1992). IL-18 is a potent inducer of IFN-, and administration of IL-18has been shown to elicit antiviral effects in VV-infected mice(Tanaka-Kataoka, M. et al. Cytokine 11, 593-599 (1999)). Recent work hassuggested that TLR3, TLR4 and TLR7 are crucial mediators of an innateimmune response to viral infection (Kurt-Jones, E. A. et al. NatureImmunol 1, 398-401 (2000); Rassa, J. C., Meyers, J. L., Zhang, Y.,Kudaravalli, R & Ross, S. Proc. Natl. Acad. Sci. USA 99, 2281-2286(2002), Alexopoulou, L., Czopik-Holt, A., Medzhitov, R. & Flavell, R.Nature 413, 696-712 (2001) and Hemmi, H. et al. Nature Immunol. 3,196-200 (2002)). Furthermore, TLR2 and TLR9 have also been implicated inresponding to some viruses (Lund, J. et al. J. Exp. Med. 198, 513-520(2003); Compton, T. et al. J. Virol. 77, 4588-4596 (2003). It ispossible that other TLRs also have a role in responding to viralinfection. If the TLR family is truly important in anti-viral hostdefense viral mechanisms to antagonise this family must exist We havefound that VV A52R is an intracellular global inhibitor of TLRsignalling. This strongly supports the emerging role of TLRs in the hostresponse to viral infection. We have found in the present invention thatdeletion of A52R from VV causes the virus to be attenuated in a murinemodel of infection (FIG. 11).

The ability of A52R to interact with both IRAK2 and TRAF6, and hencedisrupt the formation of active signalling complexes containing thesemolecules (FIGS. 3 to 6), provides a mechanistic explanation for theability of A52R to inhibit TIR-dependent signalling. A52R binds to TRAF6via its TRAF domain. This is the first demonstration of a viral proteintargeting TRAF6.

A52R is also the first viral protein identified to target IRAK2. IRAK2plays a role in many TLR pathways, including TLR3 (FIG. 61)), thereforeIRAK2 appears to play an important role in anti-viral immunity. A52Rrequires the IRAK2 death domain for association. The death domain ofIRAK2 is a protein interaction domain that allows it to associate withother proteins.

The stoichiometries of interaction strongly suggest that A52R targetsboth IRAK2 and TRAF6 independently. This apparent redundant targeting oftwo signalling molecules present on common pathways may indicate thecritical importance to the virus of inhibiting NF B activated by TLRs.However recently it has become clear that although the IL-1R/TLR familyshare a common pool of downstream signalling molecules, specificmolecules are used in different contexts leading to the range ofdifferent signals that are generated by TLRs. In response to LPS, TLR4activates cytokine release from dendritic cells by a MyD88 dependentpathway, whereas NFκB activation, IFN induction and expression ofcostimulatory molecules can occur in the absence of MyD88 (Kaisho, T.,Takeuchi, O., Kawai, T., Hoshino, K. & Akira, S. J. Immunol 166,5688-5694 (2001)). Mal/TIRAP is a novel TIR containing adapter protein,which can interact with IRAK2 and which has a role in TLR4 and TLR2signalling (Fitzgerald, K. A. et al. Nature 413, 78-83 (2001); Horng,T., Barton, G. M. & Medzhitov, R. Nature Immunol. 2, 835-841 (2001);Yamamoto, M. et al. Nature 420, 324-329 (2007); Horng, T. et al. Nature420, 329-333 (2002)). Consistent with the targeting of IRAK2 bit A52R,signalling triggered by Mal was sensitive to inhibition by A52R (seeFIG. 6 c). Significantly, the MyD88-independent pathway, which involvesthe novel TIR adapter TRIF and leads to the activation of IFN-regulatoryfactor 3 (IRF3) and induction of IFNβ, was also blocked by both A52R andΔA52R (described below).

The ability of A52R to target both IRAK2 and TRAF6 significantlyincreases the range of TIR activated signaling pathways that VV is ableto inhibit.

Surprisingly we found in the present invention that a truncated versionof A52R, ΔA52R was also potent inhibitor of IL1R/TLR superfamilysignalling. ΔA52R was generated by PCR of the portion of the A52R geneencoding amino acids 1-144, which led to a truncated version of A52Rlacking amino acids VDWRNEKLFSRWKYCLRAIKLFINDHMLDKIKSILQNRLVYVEMS at theC-terminal

It was found that ΔA52R does not target TRAF6, yet potently blocks TLRsignalling. This indicates that the interaction with IRAK2 is morecrucial for inhibition. The MyD88-independent pathway was also blockedby ΔA52R.

The present invention also provides a recombinant vaccinia virus inwhich the gene sequence of A52R is deleted. This led to an attenuationof the virus, in that when mice were infected intranasally, the deletionmutant caused reduced weight loss (FIG. 11 a) and milder signs ofillness (FIG. 11 b) compared to controls.

Live vaccinia virus is currently used as the vaccine to immunise againstand eradicate smallpox. There is a need to develop more effective andsafer smallpox vaccines due to the threat of bioterrorism. It ispossible to engineer recombinant vaccinia viruses in which vacciniagenes are deleted or altered. Deletion or alteration of vaccinia virusgenes involved in modulating the host immune response can alter theimmunogenicity and safety of a vaccinia virus for use a vaccine againstsmallpox or other orthopoxviruses, or for the development of recombinantvaccinia viruses as vaccines against other infectious diseases andcancer. Such recombinant vaccinia viruses can be engineered in whichgenes derived from other organisms are inserted (Macket, M. & Smith, G.L. J. Gen. Virol. 67, 2067-2082 (1986). The recombinant viruses retaintheir infectivity and express any inserted genes during the normalreplicative cycle of the virus. Immunisation of animals with recombinantviruses containing foreign genes has resulted in specific immuneresponses against the protein(s) expressed by the vaccinia virus,including those protein(s) expressed by the foreign gene(s) and inseveral cases has conferred protection against the pathogenic organismfrom which the foreign gene was derived. Recombinant vaccinia viruseshave, therefore, potential application as new live vaccines in human orveterinary medicine.

The present invention also provides a vaccinia virus wherein 95.2% ofthe nucleotide sequence encoding A52R is deleted (Example 6). Alterationor deletion of A52R from the vaccinia genome may increase virus safetyand immunogenicity. Such a virus or a derivative virus expressing one ormore foreign antigens may have application as an improved vaccineagainst smallpox or other orthopoxvirses, or for the application ofrecombinant vaccinia viruses as vaccines against other infectiousdiseases and cancer.

The examples presented are illustrative only and various changes andmodifications within the scope of the present invention will be apparentto those skilled in the art.

EXAMPLES

Methods

Expression Plasmids. Chimeric TLR receptors CD4-TLR1, CD4-TLR2,CD4-TLR4, CD4; TLR5 and CD4-TLR6 composed of the extracellular domain ofCD4 fused to the transmembrane domain and cytosolic tail of the TLR werea generous gift from R. Medzhitov, (Yale University, New Haven, Conn.).TLR3 was a kindly provided by K. Fitzgerald and D. Golenbock (Universityof Massachusetts Medical School. Worcester, Mass.). AU1-MyD88, Myc-IRAK2and Myc-ki expression vectors were a kind gift from M. Muzio (Muzio, M.,Ni, S., Feng, P. & Dixit, V. M. Science 278, 1612-1615 (1997)). IRAK,Flag-TRAF6, Flag-TRAF6 domain (amino acids 289-522), Flag-TRAF2expression plasmids and the mammalian expression vector pRK5 were kindlyprovided by Tularik Inc. (San Francisco. Calif.). Flag-TAK-1, andHA-TAB-1 expression plasmids were a gift from H. Sakurai (Tanabe SeiyakuCo., Osaka, Japan). Flag-TRIF was from S.Akira (Research Institute forMicrobial Diseases, Osaka University, Japan). HA-Mal expression plasmidhas been previously described (Fitzgerald, K. A. et al. Nature 413,78-83 (2001)).

Cloning of A52R and ΔA52R

The name A52R is based on the standard VV nomenclature of the Copenhagenstrain (Goebel, S. J et al, (1990) Virology 179, 247-266). A52R wascloned from the laboratory VV strain WR where it was previously calledSalF15R (Smith, G. L et al (1991) J. Gen Virol, 72 1349-1376), into themammalian expression vector pRK5. Any other suitable mammalianexpression vector such as pcDNA3.1 (available from Invitrogen) orpEF-BOS (Mizushima et al Nucleic Acids Res. 18, 5322 (1990)) for examplemay also be used.

The VV ORF A52R SalF15R in Western Reserve (WR) strain (Smith et al1991), was cloned by PCR amplification from WR DNA with primersincorporating restriction sites for EcoRI upstream and HindIIIdownstream of the ORFs. The primers used for SalF25R were5′-CGTGAATTCGTGATCACCATGGAC (sense) and 5-CGCAAGCTTCTATGACATITCCAC(antisense). The restriction sites and start and stop codons areunderlined. The resulting EcoRI-HindIII fragment was ligated into themultiple cloning site of the mammalian expression vector pRK5. Forimmunoblot analysis, epitope-tagged A52R expression vector wasconstructed, employing the same strategy, except that the 8-amino acidFlag coding sequence was inserted into the antisense primer 5′ of thestop codon.

ΔA52R encoding amino acids 1-144 of A52R was generated by PCR from fulllength A5-R and cloned into pRK5, which led to a truncated version ofA52R lacking amino acids VDVERNEKLFSRWKYCLRAIKLFINDHMLDKIKSILQNRLVYVEMSfrom the C-terminal end.

Antibodies. Polyclonal antibodies were raised against a purified,bacterially expressed glutathione S-transferase (GST) fusion of A52R,encoded by a plasmid synthesised by inserting full length A52Rdownstream of GST in the bacterial expression vector GEX4T2. Otherantibodies used were Anti-flag M2 monoclonal antibody, anti-flag M2conjugated agarose, anti-myc monoclonal antibody clone 9E10 (all fromSigma), anti-AU1 monoclonal antibody (BabCO), anti-HA polyclonalantibody (YT-1), and anti-TRAF6 (H-274) (both from Santa CruzBiotechnology). Anti-IRAK antibody was a gift from IC Ray(GlazoSmithKline, Stevenage, United Kingdom).

Cell Culture HEK 293, HEK 293T and RAW264.7 cells were cultured inDulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum(FCS), supplemented with 100 units/ml penicillin, 100 mg/mlstreptomycin, and 2 mM L-glutamine.

Example 1 A52R Inhibits Signalling by Multiple TLRs

Chimeric versions of the TLRs, comprising the murine CD4 extracellulardomain fused to the cytoplasmic domain of a given human TLR have proveduseful in probing TLR signalling pathways (Hayashi, F. et al. Nature410, 1099-1103 (2001); Ozinsky, A. et al. Proc. Natl. Acad. Sci. USA 97,13766-13771 (2000); Medzhitov, R., Preston-Hurlburt, P. & Janeway, C. A.Jr. Nature 388, 394-397 (1997)). The extracellular domain of CD4promotes homodimerisation of the molecules. Chimeras composed of theextracellular domain of CD4 fused to the intracellular domain of TLR4are constitutively active, in that overexpression of CD4-TLR4 induces NFB activation and gene induction (Medzhitov, R., Preston-Hurlburt, P. &Janeway, C. A. Jr. Nature 388, 394-397 (1997)). Using this approach forother TLRs, it was shown that some TLR cytoplasmic domains can inducegene expression as homodimers (TLR4 and TLR5), while others require apartner for this and therefore signal as heterodimers (TLR1, TLR2 andTLR6) (Hayashi, F. et al. Nature 410, 1099-1103 (2001); Ozinsky, A. etal. Proc. Natl. Acad. Sci. USA 97, 13766-13771 (2000)). Thus thesechimeras allow one to look at TLR signalling in the absence of exogenousactivator.

HEK 293 cells (2×10⁴ cells per well) were seeded into 96-well plates andtransfected the next day with expression vectors, κB-luciferase reportergene and Renilla-luciferase internal control as previously described(Fitzgerald, K. A. et al. Nature 413, 79-83 (2001)). Genejuice (Novagen)was used for transient transfections, according to the manufacturer'sinstructions. The total amount of DNA per transfection was kept constantat 220 ng by addition of pcDNA3.1 (Stratagene). 293 cells weretransfected with constitutively active CD4-TLRs (50 ng TLR4 or TLR5, or25 ng each of TLR2 & TLR6 or TLR2 & TLR1) in the presence of 80 ng emptyvector (EV) or plasmid encoding A52R, together with NFκB reporterplasmid (FIG. 1(a)). Cells were transfected with empty vector (EV), 0.5ng TLR3 or 0.5 ng TLR3 plus 150 ng A52R. Six hours prior to harvestingcells were stimulated with 0-25 g/ml poly(I:C) (FIG. 1(b)). Cells weretransfected with 0.5 ng TLR3 and stimulated with 25 g/ml poly(I:C) whereindicated (+), in the presence of increasing amounts of A52R (20-150 ng)(FIG. 1(c)).

After 24 h the cells were harvested and the reporter gene activity wasmeasured (Fitzgerald, K. A. et al. Nature 413, 78-83 (2001)). Data isexpressed as mean fold induction ±s.d. relative to control levels, for arepresentative experiment from a minimum of three separate experiments,each performed in triplicate.

Overexpression of either CD4-TLR4 or CD4-TLR5 in HEK293 cells led toinduction of an NFκB-dependent reporter gene, whereas CD4-TLR6 andCD4-TLR1 were only active when coexpressed with CD4-TLR2, to enable theformation of heterodimers (FIG. 1 a and not shown). The activation ofNFκB was in all cases inhibited by coexpression with A52R.

DsRNA is a molecular pattern associated with viral infection, and TLR3has been shown to sensitise cells to activation bypolyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue(Alexopoulou, L., Czopik-Holt, A., Medzhitov, R. & Flavell, R. Nature413, 696-712 (2001)). The effect of A52R on TLR3-dependent NFκBactivation induced by poly(I:C) was also tested. Transfection of HEK293cells with TLR3 led to strong dose-dependent activation of NFκB bypoly(I:C), which was not seen in the absence of TLR3 (FIG. 1 b). ThisTLR3-dependent induction of NFκB was completely blocked by A52R (FIG. 1b) in a dose-dependent manner (FIG. 1 c). Thus A52R is a globalinhibitor of signalling by the TLR family, with TLR3 being particularlysensitive.

A52R was also tested against TLR agonists in the murine macrophage cellline RAW264.7. Cells (2×10⁵ cells/ml) were seeded into 96 well platesand transfected the next day with either empty vector (EV) or A52R,together with NFκB luciferase reporter gene (FIG. 2 a) or an IFN-βpromoter reporter (FIG. 2 b) and Renilla luciferase internal control,using GeneJuice™ as described above. The total amount of DNA pertransfection was kept constant at 200 ng by addition of pcDNA 3.1(Stratagene). Six hours prior to harvesting cells were stimulated withthe TLR agonists 25 μg/ml Poly(I:C) (TLR3), 1 nM Pam₃CSK₄ (TLR 2) and100 ng/ml LPS (TLR4). Data is expressed as mean fold induction +/−s.d.relative to control levels, for a single experiment performed intriplicate.

Each TLR agonist led to induction of the NF % B reporter gene while theIFN-β promoter was induced by only Poly(I:C) and UPS. The activation ofNFκB and IFN-β promoter was in all cases inhibited by coexpression withA52RP Therefore A52R could inhibit signals mediated by both MyD88/Mal(e.g. LPS and Pam₃CSK₄ induced NFκB) and TRIF (e.g. Poly(I:C) inducedIFN-β promoter).

Example 2 Immunoprecipitation of A52R with TRAF6 and IRAK2

The activation of NFκB by different TLRs is mediated by a common set ofsignalling molecules. The ability of A52R to inhibit NFκB activation bymultiple TLRs suggested that its effects may be due to its interactionwith a molecule whose function is critical to signalling by the entirefamily of receptors. To test this the ability of A52R to interact withcharacterised mediators of signalling of the TLR family was examined.Flag-tagged or untagged versions of A52R were expressed in HEK 293Tcells along with lagged versions of MyD88, Mal, RAIL, TRAF6 and TAK1, oruntagged IRAK. To isolate complexes, immunoprecipitations were carriedout using antibodies directed against A52R.

HEK 293T cells were seeded into 100 mm dishes (1.5×10⁶) 24 hrs prior totransfection. Transfections were carried out using FuGENE 6 (Roche)according to manufacturers instructions. For co-immunoprecipitations, 4g of each construct was transfected. Where only one construct wasexpressed the total amount of DNA (8 g) was kept constant bysupplementation with vector DNA. Cells were harvested 24 hrs posttransfection in 750 l of lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl,1 mM EDTA, 10% glycerol, 0.5% NP40 containing 1 mM PMSF and proteaseinhibitor cocktail (1/100) (Sigma), and 1 mM sodium orthovanadate). Forimmunoprecipitation the indicated antibodies were precoupled to eitherprotein A sepharose or protein G sepharose (anti-AU1) for 1 hr at 4° C.,washed, and then incubated with the cell lysates for 2 hrs at 4° C. Theimmune complexes were washed twice with lysis buffer and once with lysisbuffer without NP40 and glycerol. Associated proteins were eluted fromthe beads by boiling in 35 l of 3×SPB (final concentrations in sample:62.5 mM Tris, 2% (w/v) SDS, 10% v/v glycerol, 0.1% (w/v) bromophenolblue)). The immune complexes were analyzed by SDS PAGE. 30 l of theimmune complex was immunoblotted for co-precipitating protein and theremaining 5 l was blotted directly for the protein directly recognisedby the immunoprecipitating antibody. For immunoblotting, primaryantibodies were detected using horseradish peroxidase conjugatedsecondary antibodies, followed by enhanced chemiluminescence (Amersham).

The results are shown in FIG. 3, where in each panel lanes 1-3correspond to lysates directly blotted for expression of the signallingmolecule, lane 4 corresponds to immunoprecipitation using antibodytowards the given signalling molecule, and lanes 5 and 6 correspond tolysates immunoprecipitated with antibody directed towards A52R andblotted for the presence of the associated signalling molecule. In eachcase the same result was obtained by immunoprecipitation in the oppositedirection, i.e. by immunoprecipitation with an antibody directed againstthe corresponding signaling molecule and blotting for A52R (not shown).

No complex formation was detected when A52R was coexpressed with MyD88(FIG. 3 a), Mal (FIG. 3 b) or IRAK (FIG. 3 c). Under the same conditionscomplexes were detected between MyD88 and IRAK2, Mal and IRAK2, and IRAKand TRAF6 (not shown), thus showing that all the constructs werefunctional. However upon expression of A52R with IRAK2 a clear complexof A52R with IRAK2 was able to be immunoprecipitated, using eitherantibodies to A52R or to IRAK2 (FIG. 3 d, compare lanes 5 and 6, and notshown). The next signalling mediator which has been positioneddownstream of the IRAK family is TRAF6. Similar to A52R and IRAK2,coexpression of A52R with TRAF6 resulted in the formation of a complexwith high stoichiometry, detected by immunoprecipitation with anantibody to either A52R or TRAF6 (FIG. 2 e, compare lanes 5 and 6, andnot shown). TRAF6 is responsible for activating TAK1 which forms acomplex with its two coactivators TAB1 and TAB2 (Wang, C. et al. Nature412, 346-351 (2001)). A52R was coexpressed with either TAK1 or TAB1 todetermine if it associates with either of these downstream mediators ofTRAF6 signalling. A weak but reproducible interaction was detectedbetween A52R and TAK1 (FIG. 3 f, compare lanes 5 and 6). No interactionwas detectable between A52R and TAB1 (not shown). These results indicatethat A52R is capable of interacting with high stoichiometry with bothIRAK2 and TRAF6. The low stoichiometry of the interaction with TAK1would suggest that this interaction is mediated by the binding of A52Rto endogenous TRAF6.

The specificity and functional consequences of the interaction of A52Rwith TRAF6-containing complexes were examined. FIG. 4 a shows that A52Rcould be immunoprecipitated with endogenous TRAF6 (compare lanes 3 and4, top panel). To determine the regions in TRAF6 responsible forinteracting with A52R, truncated versions of TRAF6 were co-expressedwith A52R and tested for their ability to interact byimmunoprecipitation. A truncated version of TRAF6 composed of just theTRAF domain was able to interact with ASR to the same extent as the fulllength TRAF6 (FIG. 4 b, lanes 5 and 6, compare top and middle panels).Thus these results show that A52R interacts with the TRAF domain ofTRAF6. To test the specificity of A52 for TRAF6, A52R was coexpressedwith Flag-tagged TRAF2, and the ability to form a complex was monitored.Using identical conditions to where a TRAF6 interaction was detected(FIG. 4 b top panel), no interaction between A52R and TRAF2 was detectedby immunoprecipitation using either an A52R antibody (FIG. 4 b lowerpanel) or a Flag antibody (not shown).

Example 3 Disruption of a TRAF6-TAB1-Containing Signalling Complex byA52R

The effect of A52R on the ability of TRAF6 to form active signallingcomplexes necessary for the activation of NFκB was assessed. TAK1 andits coactivators TAB1 and TAB2 are downstream targets of TRAF6 that areimportant in NFκB activation (Wang, C. et al. Nature 412, 346-351(2001)). We detected a TRAF6-TAB1-containing complex by IP (FIG. 5, toppanel, lane 2), and examined the effect of A52R on the formation of thiscomplex. Increasing amounts of plasmid encoding A52R-Flag werecotransfected into 293T cells along with a constant amount of plasmid (2g) encoding Flag-TRAF6 and HA-TAB1. The total amour-t of DNA was keptconstant in each sample using empty vector. The amount ofTAB1-containing complex formed was assessed by immunoprecipitation usinganti-Flag antibody, followed by western blotting with anti-HA antibody.As the expression of A52R increased, the amount of TAB able to beco-immunoprecipitated with TRAF6 decreased steadily (FIG. 5, top panel).Equal expression of TRAF6 and TAB1 was confirmed by direct immunoblot(not shown). This effect was specific to TRAF6 as the expression ofincreasing levels of A52R bad no effect on the formation of a TAB1-TAK1complex (FIG. 5, lower panel).

Example 4 A52R Inhibition of Mal-Induced NFκB Activation, and theDissociation of a Mal-IRAK2 Complex

(i) A52R Requires the Death Domain of IRAK2 for Interaction

The specificity and functional consequences of the interaction betweenA52R and IRAK2 containing complexes was examined. To determine theregions in IRAK2 responsible for interacting with A52R, truncatedversions of IRAK2 were coexpressed with A52R and tested for theirability to interact by IP. 293T cells were cotransfected with flag-A52R(4 g) a n d either 4 g of myc-IRAK2, or myc-kIRAK2 (a variant of thedeath domain containing residues 97-590). Lysates were prepared 24 hlater and flag-A52R was immunoprecipitated with anti-flag antibody andblotted with anti-myc to detect the presence of IRAK2 or kIRAK2 (FIG. 6a upper panel). Immunoprecipitation and immunoblot of lysates (1/7 ofimmunoprecipitation) with anti-flag antibody demonstrated equalefficiency of immunoprecipitation and A52R expression (FIG. 6 a middlepanel). Lysates were also blotted with anti-myc antibody to monitor theexpression of IRAK2 and kIRAK2 (FIG. 6 a lower-panel). kIRAK2 lacks thedeath domain was unable to interact with A52R (FIG. 6 a, top panel,compare lanes 3 and 4). Thus A52R requires the death domain in order tointeract with IRAK2.

(ii) Role of IRAK2 in Inhibition of TLR-Induced NFκB Activation by A52R

293 cells were transfected with constitutively active CD4-TLRs (50 ngTLR4 or TLR5, or 25 ng each of TLR2 & TLR6 or TLR2 & TLR1) in thepresence of 80 ng empty vector (EV) or plasmid encoding A52R, togetherwith NF B reporter plasmid (FIG. 6 b, upper graph). In FIG. 6 b lowergraph, 293 cells were transfected with 0.5 ng TLR3 and stimulated with25 g/ml poly(I:C) where indicated (+), in the presence of increasingamounts of ΔIRAK2 (5-80 ng). After 24 h the cells were harvested and thereporter gene activity was measured (Fitzgerald, K. A. et al. Nature413, 783 (2001)). Data are expressed as mean fold induction ±s.d.relative to control levels, for a representative experiment from aminimum of three separate experiments, each performed in triplicate.Similar to TRAF6, there was a correlation between inhibition ofTLR-induced NFκB activation by A52R, and a role for IRAK2 in thesepathways: FIG. 61 b shows that each CD4-TLR induced signal that wassensitive to A52R was also blocked by dominant negative IRAK2 (uppergraph). It was also shown that IRAK2 has a role in TLR3-dependentpoly(I:C)-induced NFκB activation, since dominant negative IRAK2 led toa dose-dependent inhibition of this signal (FIG. 6 b, lower graph). ThusIRAK2 has a wide-ranging role in many TLR pathways to NF % B activation,providing a further rationale for the inhibitory effect of A52R on TLRsignalling.

(iii) A52R Inhibits Mal-Induced NF B Activation, and Disrupts aMal-IRAK2 Complex

Activation of NFκB by NFκB, an adapter protein which acts downstream ofTLR4 and TLR2, may be mediated via its binding to IRAK2 (Fitzgerald, K.A. et al. Nature 413, 78-83 (2001)). Given that IRAK2 is a target forA52R, the effect of A52R on the ability of Mal to activate NFκB wasexamined. In FIG. 6 c, 293 cells were transfected with 10 ng Mal whereindicated (+), in the presence of increasing amounts of A52R (5-80 ng),together with NF B reporter plasmid. After 24 h the cells were harvestedand the reporter gene activity was measured (Fitzgerald, K. A. et al.Nature 413, 78-83 (2001)). Data are expressed as mean fold induction±s.d. relative to control levels, for a representative experiment from aminimum of three separate experiments, each performed in triplicate.Overexpression of Mal was able to activate NF % B and this activationwas clearly inhibited by the coexpression of A52R in a dose-dependentmanner (FIG. 6 c). The effect of A52R expression on the ability of Malto interact with IRAK2 was also examined. Increasing amounts of plasmidencoding A52R-Flag were cotransfected into 293T cells along with aconstant amount of plasmid encoding myc-IRAK2 (2 g) and HA-Mal (2 g).Lysates were prepared after 24 hrs, and the amount of IRAK2-Mal complexformed was assessed by immunoprecipitation using anti-HA antibody,followed by western blotting with anti-myc antibody as the expression ofA52R increased, the amount of IRAK2 able to be coimmunoprecipitated withMal decreased steadily (FIG. 6 d). This decrease in complex formationwas not due to a decrease in the expression of either IRAK2 or Mal sincedirect immunoblot showed equal expression of both signalling moleculesas the expression of A52R increased (not shown). These results show thatA52R is able to inhibit the activation of NF B by Mal, and thisinhibition correlates with dissociation of an active Mal-IRAK2signalling complex upon increasing A52R expression.

Example 5 ΔA52R, a Truncated Version of A52R, is a Potent Inhibitor ofTLR Signalling

(i) ΔA52R co-IPs with IRAK2 But not TRAF6

In order to begin to map the sites of interaction between A52R and TRAF6and IRAK2, a truncated version of A52R lacking 46 amino acids at theC-terminal was generated. ΔA52R was first tested for its ability to bindIRAK2 and TRAF6. HEK293T cells were seeded into 100 mm dishes 24 hrsprior to transfection with GeneJuice™, as described in Example 2. Asbefore, 4 ii of each construct was used, and cells were harvested andlysed after 24 μl.

The results are shown in FIG. 7. In FIG. 7 a, A52R is clearly seen to becapable of interacting with IRAK2 (as seen by a band in lane 6 but notin lane 4), as was the case for A52R (see FIG. 3 d). However unlikeA52R, an association with TRAF6 could not be detected for ΔA52R. This isseen in FIG. 7 b, whereby coIP with an anti-A52R antibody pulls downTRAF6 when A52R is present, but fails to do so when ΔA52R is present(compare lane 3 top panel where a band corresponding to TRAF6 isapparent above the antibody heavy chain band, to lane 6 top panel wherethere is no such band above the heavy chain).

(ii) Like A52R, ΔA52R Inhibits TLR Signalling

Given that ΔA52R can interact with IRAK2, but not detectably with TRAF6,it may provide a useful tool in order to determine the relativecontribution of the interaction of A52R with IRAK2 and TRAF6 toinhibition. Therefore the effects of ΔA52R on TLR signalling, inparallel to A52R, were examined.

FIG. 8 shows a comparison of the effect of A52R and ΔA52R on IL-1 andTLR4-dependent NFκB activation. HEK 293 cells were transfected withexpression vector for A52R and reporter genes, as described inExample 1. FIG. 8 a shows that ΔA52R was actually a slightly more potentinhibitor of IL-1 than A52R over a range of doses of plasmid. Thisheightened inhibition by ΔA52R is even more apparent for TLR4, where amore potent effect of ΔA52R compared to A52R is clearly seen at thesingle low dose of 10 ng plasmid. These results suggest that interactionof A52R with IRAK2 may be more fundamental for inhibition of TLRsignalling than interaction with TRAF6. In fact it may be that becauseΔA52R escapes interaction with TRAF6, it is able to block TLRs moreeffectively.

(iii) Inhibition of TRIF Induced Signalling by A52R and ΔA52R

Activation of IRF3 by TLR3 and TLR4, which leads to IFNβ induction, ismediated through the adapter molecule TRIF (see ‘Background’). Theresults from FIG. 2 b suggested that A52R could inhibit theTRIF-dependent pathways to INFβ for TLR3 and TLR4. Here, the directeffect of A52R and ΔA52R on signals activated by the over-expression ofTRIF was determined. HEK 293 cells were transfected with 10 ng TRIFwhere indicated (+), in the presence of 100 ng of plasmid encoding A52Ror ΔA52R, together with an NF % B reporter plasmid (FIG. 9 a) or anIFN-β promoter reporter plasmid (FIG. 9 b). After 24 hours the cellswere harvested and the reporter gene activity was measured (Fitzgerald,K. A. et al. Nature 413, 78-83 (2001). Data is expressed as mean foldinduction +/−s.d. relative to control levels, for a representative threeseparate experiments, each performed in triplicate. Overexpression ofTRIF led to the activation of NFκB and IFN-β. Each of these activationswas clearly inhibited by the coexpression of both A52R and ΔA52R.Clearly, ΔA52R is again capable of more potent inhibition than A52R.

(iv) Differential Effect of A52R and ΔA52R on p38 MAP Kinase Activation

Examination of the effect of A52R and ΔA52R on the induction andinhibition of p38 MAP kinase gave a clue as to why ΔA52R may be a betterTLR inhibitor. The MAP kinase p38 has been shown to be important in theinduction of genes by IL-1 and TLR agonists such as LPS. In order tomeasure the effect of A52R and ΔA52R on p38 MAP kinase, the StratagenePathdetect™ System was employed. HEK 293 cells were transfected with aRenilla-luciferase internal control and a pFR-luciferase reporterconstruct in the presence of a plasmid encoding GAL4-CHOP together withincreasing amounts (10, 30 and 100 ng) of plasmid encoding either A52Ror ΔA52R (FIG. 10 a). Surprisingly, A52R was capable of strongly drivingp38 MAP kinase activation, while ΔA52R had little stimulatory effect. Itis possible that the interaction of A52R with TRAF6 triggers p38activation, as has been shown for other TRAF6-interacting host proteinssuch as TIFA (Takatsuna, H. et al. J. Biol. Chem. 278, 12144-12150(2003).

The effect of A52R and ΔA52R on IL-1 and TLR4 mediated activation of p38was next tested. Here, cells were stimulated for 6 h with 100 ng IL-1(FIG. 10 b), or transfected with 50 ng CD4-TLR4, as in previousexperiments. Both of these treatments drove p38 activation, and in eachcase A52R had a stimulatory effect on the activity, while ΔA52R had anopposite effect and was capable of blocking activation. Thus the abilityof ΔA52R to inhibit TLR signalling more potently than A52R may berelated to its ability to inhibit p38 activation, arising from escapingTRAF6 interaction.

Example 6 Deletion of A52R Gene from VV Attenuates the Virus

The role of A52R in the V life cycle was investigated by theconstruction of a deletion mutant lacking the A52R gene and by thecomparison with wild type and revertant controls. A VV mutant lacking95.2% of the ASR gene (D-A52R) was constructed by transient dominantselection (Falkner, F. G. & Moss, B. (1991) J. Virol. 64, 3108-3111). Aplaque purified wild type virus (WT-A52R) and a revertant virus(A52R-REV) in which the A52R gene was reinserted at its natural locuswere also isolated. The virulence of the viruses was investigated in amouse intranasal model. Female, 6-week old Balb/c mice wereanaesthetized and inoculated with 10⁴ p.f.u. of VV in 20 μl ofphosphate-buffered saline. A control group was mock infected with PBS.Each day the weights of the animals and signs of illness were measuredas described previously (Alcami, A. & Smith, G. L. (1992) Cell 71,153-167). The loss of the A52R gene did not affect the replication ofthe virus in cell culture or the plaque size (data not shown). However,in a murine intranasal model the deletion mutant caused reduced weightloss (FIG. 11 a) and milder signs of illness (FIG. 1 b) compared tocontrols. Thus the A52R protein contributes to virus virulence and thisis likely to be due to the inhibition of IL1R/TLR signalling.

Taken together, these results demonstrate that A52R from VV is able toinhibit TLR-induced NUMB activation by associating with key signallingmolecules and thus disrupting the formation of active signallingcomplexes. The ability of A52R to disrupt TLR signalling has relevanceto VV virulence, since deletion of A52R attenuates the virus.

In this specification some references have been included which werepublished after the priority date of the application. These are includedfor the reader's assistance only.

The invention is not limited to the embodiments hereinbefore describedwhich may be varied in detail.

1. An orthopoxvirus vector, such as vaccinia, wherein the A52R proteinfrom vaccinia, or a closely related protein from any orthopoxvirus isnot expressed or is expressed but is non-functional.
 2. A vector asclaimed in claim 1 wherein part or all of the nucleotide sequenceencoding A52R is deleted from the viral genome.
 3. A vector as claimedin claim 1 wherein the nucleotide sequence encoding A52R is inactivatedby mutation or the insertion of foreign DNA.
 4. A vector as claimed inclaim 1 wherein the nucleotide sequence encoding A52R is changed.
 5. Avector as claimed in claim 1 wherein the A52R gene comprises amino acidSEQ ID No.
 1. 6. A vector as claimed in claim 1 having enhancedimmunogenicity and/or safety compared to the wild type orthopoxvirus. 7.A medicament comprising an orthopoxvirus vector as claimed in claim 1.8. A vaccine comprising an orthopoxvirus vector as claimed in claim 1.9. A recombinant orthopoxvirus incapable of expressing a native A52Rprotein.
 10. A vaccine comprising a recombinant virus as claimed inclaim
 9. 11. A method of attenuating an orthopoxvirus such as vacciniavirus, comprising the steps of: (a) deleting part or all of thenucleotide sequence encoding A52R from the viral genome; and/or (b)inactivating one or more of said nucleotide sequence by mutating saidnucleotide sequence or by inserting foreign DNA; and/or (c) changingsaid nucleotide sequence to alter the function of a protein productencoded by said nucleotide sequence.
 12. A method of inhibiting IL1R/TLRsuperfamily signalling comprising administering an effective amount ofvaccinia A52R protein, or a closely related protein from anyorthopoxvirus or a functional peptide, peptidometic fragment orderivative thereof or a DNA vector capable of expressing such a proteinor fragment thereof.
 13. A method of modulating anti-viral immunity in ahost comprising administering an orthopoxvirus vector as claimed inclaim 1 or a functional peptide, peptidometic, fragment or derivativethereof.
 14. An immunogen comprising an orthopoxvirus vector as claimedin claim 1 or a recombinant virus vector as claimed in claim
 9. 15. Useof a vaccinia virus A52R protein or a closely related protein from anyorthopoxvirus, or a functional peptide, peptidometic, fragment orderivative thereof, or a DNA vector expressing any of the above in themodulation and/or inhibition of IL1R/TLR superfamily signalling.
 16. Useas claimed in claim 15 in the modulation and/or inhibition of IL1R/TLRsuperfamily induced NFκB activation.
 17. Use as claimed in claim 15 inthe modulation of IL1R/TLR superfamily induced MAP kinase activation.18. Use as claimed in claim 15 in the modulation or inhibition of TLRinduced IRF3 activation.
 19. Use as claimed in claim 15 wherein thevaccinia virus A52R protein, or a closely related protein from anyorthopoxvirus, inhibits Toll-like receptor proteins.
 20. Use as claimedin claim 15 in the modulation and/or inhibition of NF-κB activity byinteraction of A52R with TRAF6.
 21. Use as claimed in claim 20 whereinthe A52R protein inhibits formation of an endogenous signalling complexcontaining TRAF6/TAB1.
 22. Use as claimed in claim 15 in the modulationand/or inhibition of NF-κB activity by interaction of A52R with IRAK2.23. Use as claimed in claim 15 wherein the A52R protein inhibitsMal/IRAK2 interaction.
 24. A viral protein comprising amino acid SEQ IDNo.
 2. 25. Use of a viral protein as claimed in claim 24 or a functionalpeptide, peptidometic, fragment or derivative thereof in the modulationand/or inhibition of IL1R/TLR superfamily signalling.
 26. Use as claimedin claim 25 in the modulation and/or inhibition of IL1R/TLR superfamilyinduced NFκB activation.
 27. Use as claimed in claim 25 in theinhibition of IL1R/TLR superfamily induced p38 MAP kinase activation.28. Use as claimed in claim 25 wherein the said truncated vaccinia virusA52R protein inhibits Toll-like receptor proteins.
 29. Use as claimed inclaim 25 in the modulation and/or inhibition of NF-κB activity byinteraction of the said truncated A52R with IRAK2.
 30. A peptide derivedfrom, and/or a small molecule inhibitor designed based on a viralprotein comprising amino acid SEQ ID No. 1 or SEQ ID No.
 2. 31. A methodof screening compounds that modulate the NF-κB and/or p38 MAP kinaserelated pathway comprising measuring the effect of a test compound onthe interaction of A52R or a viral protein fragment comprising aminoacid SEQ ID No. 2 or a functional peptide, peptidometic, fragment orderivative thereof with TRAF6 and/or IRAK2.
 32. A method of identifyingsignalling pathways that require TRAF6 and/or IRAK2, comprisingmeasuring their sensitivity to A52R or a viral protein comprising aminoacid SEQ ID No.
 2. 33. Use of a functional peptide, peptidometic, orfragment derived from vaccinia virus A52R protein, or any closelyrelated orthopoxvirus protein, or a small molecule inhibitor designedbased on A52R in the treatment and/or prophylaxis of IL-1R/TLRsuperfamily-induced NF-κB or p38 MAP kinase related diseases orconditions.
 34. Use as claimed in claim 33 wherein the NF-κB relateddisease or condition is selected from any one or more of a chronicinflammatory disease, allograft rejection, tissue damage during insultand injury, septic shock and cardiac inflammation, autoimmune disease,cystic fibrosis or any disease involving the blocking of Thl responses.35. Use as claimed in claim 34 wherein the chronic inflammatory diseaseincludes any one or more of RA, asthma or inflammatory bowel disease.36. Use as claimed in claim 34 wherein the autoimmune disease issystemic lupus erythematosus.
 37. Use as claimed in claim 33 in thetreatment and/or prophylaxis of inflammatory disease, infectious diseaseor cancer.
 38. Use as claimed in claimed in claim 33 wherein the proteinis derived from an orthopoxvirus.